...for its role in molecular biology, biotin (Vitamin H or Vitamin B7) is first an essential coenzyme that ...determined to be due to a deficiency in Vitamin H later renamed Vitamin B7/biotin. Egg white injury would plague...biotinylation. This 13 amino acid sequence is termed AviTag. Using AviTag, one can label a protein of interest with...bacterial fusion proteins Biotin is an essential vitamin for life Although biotin is most commonly known...bacteria and some fungi, thus mammals must obtain the vitamin through external sources. Humans absorb most of... biotinylate macromolecules of interest both in vitro and in vivo. Biotinylation reagents Biotin’s exposed...Scientific's Biotinylation Reagent Selection Tool. AviTag and BirA Proteins can also be biotinylated by exploiting...

...to control Rho GTPase activity. A) Schematic of Opto-RhoGEF design and activity. B) OptoTIAM (magenta)... light-sensitive ion channels used to control activities like neuronal signaling — think of a mouse with...there’s a lot more to optogenetics than neuronal activity. Researchers have designed a variety of photoswitchable...severing. A) Schematic of Opto-katanin constructs and activity. EB3N-VVDfast-iLID (anchor) and SspB-mCherry-p60...microtubule severing and has very low dark-state activity and toxicity. All of that means this light-powered...called SuperPLDs with 10, 30, 50, and 100 times the activity of the original, generated using directed evolution... with high efficiency in mammalian cells and in vitro, opening up a wide realm of possibilities. The CRY2...

...can interfere with activity. The resulting system was coined as CRISPR–Cas9 activity switching via a novel...stemmed from the goal of reducing off-target Cas9 activity. The use of nickases, prime editing, anti-CRISPR...targeting specificity or reduce the duration of Cas9 activity.  The field of optogenetics is renowned for enabling...such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR...NLSx3-CRYPHR-p65 - it had the lowest background activity in the dark state and highest fold induction at...where light is required to reconstitute enzymatic activity. Photoactivatable genome modifications by NHEJ...low background and high fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system...

...and predicted on-target activity was balanced with predicted off-target activity when selecting guides....lower off-target activity, but it could also be caused by a reduction of on-target activity. However, there...-target activity, Morgens et al drew some generalizable conclusions about off-target activity, demonstrate...off-target cutting without a reduction of on-target activity, but it’s unclear if this holds true in genome-wide...sites that lack open chromatin marks, DNase hypersensitivity, or are in an enhancer, transcription factor...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated...guides with high GC content have greater off-target activity. Short gRNAs have lower off-target cutting and...

... chance of cross-reactivity Higher level of background stainingLess sensitivityLess specificityLimited...specificityIncreased chance of cross-reactivity Sandwich High specificityHigh sensitivity Longer protocolIncreased...moleculesReduced chance of cross-reactivity Less sensitivityLess specificityLimited availability of conjugated... ELISAs for their accuracy, specificity, and sensitivity (a good ELISA can detect picogram quantities ...procedure removes one potential source of cross-reactivity.   While the method is simple and saves time,...Incorporating a secondary antibody increases the sensitivity of the assay, since multiple secondary antibodies...secondary antibodies can also be a source of cross-reactivity.  Sandwich ELISAs In the most common type of...

...between recipients.   In vitro prediction of drug immunogenicity  The goal of in vitro prediction of immunogenicity..., 2020).  Evaluating a drug’s immunogenicity in-vitro helps researchers understand a drug’s safety and...depending on the person. However, in silico and in vitro assessment of immunogenicity can help evaluate risk...silico prediction algorithms fail to capture. In vitro evaluation of ‌therapeutic immunogenicity is typically...induction (Groell et.al, 2018). Thus, microscopic in-vitro models can be built that contain various components...components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of therapeutic...efficacy.  Better together Combining in silico and in vitro approaches enables researchers to develop safer ...

...Recombinant Antibody Description Source Species Isotype Reactivity Recommended Applications PI Do you have suggestions...

...Recombinant Antibody Description Source Species Isotype Reactivity Recommended Applications PI Do you have suggestions...

... light-activated tool for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated...to blue light, this channel activates neuronal activity by opening and allowing positively charged ions...developed many optogenetic tools to control cellular activity with exquisite precision using only light. Optogenetics... tools, can control everything from electrical activity to gene expression, and even CRISPR based gene... optogenetic tool causing a change in cellular activity or gene expression. Experimental output is measured...(rhodopsins) either excite or inhibit neuronal activity upon exposure to light within a specific range...downstream signaling cascades (reviewed in Abreu & Levitz, 2020), including light-sensitive chimeric GPCRS...

...amino acid) AviTag sequence in a protein and adds biotin to a lysine residue within the AviTag. However,...modification of the purified target protein with the vitamin biotin. The interaction between biotin and the .... Avi-Tag plasmid for improved biotinylation selectivity A common strategy to biotinylate proteins involves... cannot be well-controlled. The lack of site selectivity typically diminishes the apparent affinity for...relatively low efficiency of biotinylation, at least in vitro. Our solution was to exploit the best of both worlds...carboxyl-terminus of the fusion protein includes an AviTag and a 6X HisTag for affinity purification (see ...ligase will biotinylate the lysine residue in the AviTag. As a side note, we found that adding a TEV linker...

...mammalian cell expression and a T7 promoter for in vitro transcription of RNA. TALENs expressed from these...

...CRISPR systems with wiki entries describing enzyme activity, experimental considerations, and more, created...

...CRISPR systems with wiki entries describing enzyme activity, experimental considerations, and more, created...

...CRISPR systems with wiki entries describing enzyme activity, experimental considerations, and more, created...

...CRISPR systems with wiki entries describing enzyme activity, experimental considerations, and more, created...

...alpha2-AR. Selectivity Norepinephrine and dopamine are similar molecules, and cross-reactivity is a very...without biosensors to directly visualize their activity. In 2019, the first generation of norepinephrine...But this first generation of tools had limited sensitivity and speed and were only available as green probes...Kagiampaki et al. found nLight is suitable for in vitro, ex vivo, and in vivo experiments, and nLightG was... norepinephrine uptake throughout the brain. Sensitivity In ex vivo studies on mouse brain slices, nLightG... and nLightR showing significantly increased selectivity for norepinephrine over dopamine. All three sensors...to high concentrations of dopamine. This cross reactivity will need to be accounted for in your experimental...

...are critical: (1) connectivity to other data sources used in research; (2) connectivity to other tools used... each of these kinds of connectivity facilitates reproducibility. Connectivity to other data sources The...ecosystems and act as data silos because they limit connectivity with other tools and resources. Thus, it’s difficult... used in research; and (3) connectivity to open source data repositories. To illustrate this, let’s take...principles and has emerged as a byproduct of connectivity. Between 2012 and 2017 RSpace was developed ...have been implemented by providing support for connectivity to external data sources, tools used in research...data stored on institutional and lab servers. Connectivity to other tools used in research Just as researchers...

... the video below for an example of two-photon intravital microscopy from PLoS Pathogens - Kamenyeva et... technique and the in vivo live-cell imaging (intravital imaging) studies it’s enabled are revealing the...rapidly than any other cell type in the body! Intravital imaging powered by two-photon microscopy has ...of pathogen-immune cell interaction relied on in vitro studies where it was difficult to apprehend the ...nodes, brain, liver, gut and skin. Two-photon intravital microscopy. The video above depicts the infiltration...Technical challenges of two photon microscopy Intravital imaging is an easy way to understand what is ...fluorescence) are also useful tools to use with intravital imaging. These tools allow one to study the kinetics...

...CRISPR systems with wiki entries describing enzyme activity, experimental considerations, and more, created...

...construction of RAS pathway gene expression vectors for in vitro and in vivo research. TP53 Mutagenesis Library :...